Systems and methods for detection of analytes in biological fluids

ABSTRACT

The invention provides a heterogeneous immunoassay for detection of antibodies and antigens based on specific antigen-antibody immune complex formation with multiple antigen-bearing conjugate components. The invention further provides means for optimizing the assay format for the detection of both low and high-affinity antibodies, and provides means for quantitative detection of both antibody and the corresponding antigen present in a sample.

RELATED U.S. APPLICATION

This application is a divisional application of U.S. Ser. No.10/138,654, filed May 3, 2002, which is hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to an immunochemical assay and method forqualitative or quantitative detection of analytes in biological fluids.

BACKGROUND ART

Antibodies are protein molecules which specifically bind antigens thatinitiate the formation of the antibodies. Antibodies are produced byB-lymphocytes and plasma cells, and are present in blood plasma (assecretory antibodies or cell bound), lymphatic fluids, cerebrospinalfluid, mucus and other extracellular secretions (e.g., saliva), and insome instances, urine. Assays for antibodies have been widely applied tothe examination and diagnosis of many infections, and autoimmune andallergic conditions.

There are currently several known immunoglobulin classes, IgG, IgM, IgA,IgE and IgD. These immunoglobulins are involved in different types ofimmune responses, and their involvements are dependent, for example, onantigen structures, type of organisms which bear these antigens, timeafter immunization, and presence of other antigenic molecules in themicroorganisms. It is believed that antibody classes important forserological testing and secretory immunoglobulins contain two or moreidentical antigen-combining sites. In particular, it is believed thatthere are two sites for IgG and IgE, four for IgA, and up to 10 for IgM.This mean that at least two antigen molecules or specific antigenic(epitopic) substances can bind simultaneously to one antibody molecule.

It should be noted that affinity of antibodies to specific antigens maynot be constant. Beside the spectrum of polyclonal antibodies which candiffer in affinity at varying stages of an immune response depending onthe particular pathogen/immunogen, affinity of antibodies can changewith time, from low to high affinity, for instance, during the antibodymaturation process. Moreover, several antibody classes cansimultaneously recognize the same antigen. In the case of bacterial,viral, protozoan, parasitic, or fungal infection, the IgM class canappear at the early stages of an immune response, usually as antibodywith a low affinity constant, while the IgG class can appear later inthe immune response. For serological testing constant, while the IgGclass can appear later in the immune response. For serological testingof many infections, the ability to detect antibodies at the earlieststages of seroconversion can be very important in determining atreatment. Because the IgM class antibodies often appear during theearly stages of infection, their presence is often considered as aserological indication of early stage of infection.

Antibodies can be detected using a variety of immunoassay protocols.Among conventional methods, the most widely used works on principles ofsolid phase immunoassay using immobilized antigens and secondaryclass-specific anti-immunoglobulins conjugated with detector labels,such as, enzyme, fluorophores, chemiluminescent, metal sols, dyedpolymeric particles, or labeled proteins which bind to immunoglobulins(protein A and G). A disadvantage of such a method is that a falselypositive result can be obtained due to non-specific and cross-reactivereactions related to simple adsorption of non-specific antibodies oncoated solid phase, or the presence of unwanted antigenic substances inantigens used for adsorption on solid phase. This can happen even incase of using relatively pure antigens obtained by methods ofrecombinant technology. Moreover, the presence of, for instance,autoantibodies, rheumatoid factor, or polyspecific/polyreactiveantibodies, can lead to false results. Even in case of using veryspecific small peptide antigens, false positives can arise as result ofcontaminations in the reagents used for immobilization on solid phase.

In order to reduce the occurrence of false positives, steps, such as,selection of sample dilution, incubation time, temperature, and diluentsused, have been taken to reduce potential cross-reactive reactions.However, such steps can require the application of many serologicaltests and can reduce detection sensitivity. For example, in a solidphase immunoassay using solid phase immobilized antigens, the presenceof conjugates antigen-specific antibodies with detector label cancompete with antibodies in a sample to be detected to bind with theimmobilized antigens. As a result, although this competitive test isrelatively simple and may be less susceptible to non-specific reactions,its sensitivity for detection can be relatively low.

In another immunoassay for specific antibody detection based oncompetitive binding, a specific antibody may be immobilized in a solidphase. However, during incubation, labeled antigens in the sample andthe antibody to be detected in the sample can compete to bind with theimmobilized antibody. As with the previous example, sensitivity ofdetection may be affected due to competitive binding, and as a result,may require a significant amount of pure anti-antigen antibodies foruse.

In another immunoassay, the solid phase may be coated with a classspecific immunoglobulins. Labeled antigens may be used in the sample ofthe antibody to be detected to form an immune complex with the classspecific immunoglobulins in the solid phase. One disadvantage of thisimmunoassay is the limited binding capacity of the class specificimmunoglobulins in the solid phase, which can limit the sensitivity ofspecific antibody detection.

A further variation of solid phase immunoassay involves the use of solidphase coated antigens to capture specific antibody and subsequentdetection of captured/bound antibody with a labeled antigen. However, aprozoning phenomenon may be a problem for this approach. In particular,the density of antigen on solid phase should be carefully optimized toprotect the antibody from binding with the solid phase antigen throughall combining sites (e.g., IgG class) on the antibody, which reducenumber of sites available on the antibody to bind with labeled antigen.A second incubation with labeled antigen is described in U.S. Pat. No.6,121,006 as a way for increasing sensitivity of this type of assay.

U.S. Pat. No. 6,030,770 discloses a method for antibody detection usinglabeled antigen and a solid phase capture system. In this method, thesolid phase comprises immobilized antigens and anti-immunoglobulinantibodies introduced into the capture system through an additionalbridge. The bridge, as disclosed, includes analyte-specific antibodiescapable of recognizing ligand-labeled anti-species class specificantibodies.

U.S. Pat. No. 4,778,751 discloses a method for detection of circulatingantibodies, which method is based on the use of a ligand-labeledpolymeric matrix. In this method, the matrix is conjugated with multipleantigen molecules, a ligand-binding partner immobilized on solid phase,and anti-immunoglobuline detector reagent.

In U.S. Pat. No. 4,945,042, a three reagents antibody detection systemis disclosed. The detection system, as disclosed, comprises twocomponents of a specific binding pair. One component is included in thecapture system as a conjugate of StrAv with thermo polymerized BSA ofanti-immunoglobulin antibodies, while the second component is conjugatedto the antigen. A third reagent is labeled and is a conjugate of theantigen.

U.S. Pat. No. 5,236,849 discloses a detection method utilizing anantigen containing simultaneously two different labels belonging to twoaffinity pairs, each with its counterparts distributed between separatesolid phases. This approach permits for significant reduction ofnon-specific reaction by using immune-complex transfer(dissociation-recapture) for detection in a second container.

Lyphophilic bridges, such as liposomes or some other reversible bridge,are disclosed in U.S. Pat. Nos. 5,312,730 and 5,705,338 as a way todissociate an immune complex from a first solid phase to permit transferinto a second solid phase for detection.

Small peptide antigens, containing specific epitopes, mono or limitedamount, can be powerful instrument for development of highly specificserological test. The role of these peptides, whether obtained bychemical synthesis or recombinant technology, have become increasinglyimportant. However, affinity of antibodies against these small epitopesis sometimes lower than the affinity to a sequence in whole protein.Moreover, affinity of antibodies to selected peptide antigens can varydue to the presence of various strains of pathogens and its geneticvariability. Accordingly, it is desirable to provide simple methods,which can permit the detection of antibodies that may have low affinityto selected epitopes.

SUMMARY OF INVENTION

The present invention provides, in one embodiment, a simpleheterogeneous immunoassay for detection of antibodies based on specificantigen-antibody immune complex formation with dually labeled antigen.

In another embodiment, the present invention provides methods fordetection of antibodies against peptide epitopes and haptens ordetection of other analytes with multiple identical binding epitopes. Inthis manner, the method of the present invention can permit detectionof, for example, multiple antibody classes in one sample, includingantibody with a low affinity constant.

In another embodiment, the present invention provides a simplifiedmethod for sample preparation, thereby allowing the assays being carriedout to be insensitive to sample dilution, while eliminating factorswhich can lead to various non-specific reactions and false positivesfrequently seen in conventional serological tests usinganti-immunoglobulin conjugates.

The method of the present invention can be used in a variety of assayformats, including ELISA, dot-blot, flow-through (e.g.,immunoconcentration and immunofiltration), and lateral flow, includingthe possibility for quantitative determination.

In accordance with one embodiment, the present provides a method fordetecting an antibody that binds to an antigen or hapten in a sample.The method includes providing a first component having a ligand linkedto a solid phase adsorbing composition through a chemical spacer. In anembodiment, the ligand may be biotin, the solid phase adsorbingcomposition may be albumin and the spacer may be polyethylene glycol.Also provided is a second component having a ligand-binding moiety andthe antigen or hapten that binds the antibody. The ligand-binding moietyin an embodiment can be avidin or streptavidin. A third component can beprovided comprising an antigen or hapten and a label. The label, in anembodiment, may be one of the following, an enzyme, a fluorescent probe,a chemiluminescent probe, a metal, a non-metal colloidal particle, apolymeric dye particle, and a pigment molecule. The method permitscontacting the first component with a solid phase, so as to immobilizethe first component. Subsequently, the antibody sample is contacted withthe first, second and third components to allow the third component toform an immune complex with the first and second components by bindingto the second component through the antibody present in the antibodysample. Thereafter, uncomplexed third component may be separated fromthe solid phase and the label present in the immune complex may bedetected. The detection of the label indicates the presence of theantibody in the antibody sample, and thus the detection of the antibodyin the sample.

The present invention also provides, in one embodiment, a composition ofmatter comprising a ligand linked to a solid phase adsorbing compositionthrough a chemical spacer. In an embodiment, the ligand may be biotin,the solid phase adsorbing composition may be albumin and the spacer maybe polyethylene glycol.

The present invention further provides, in an embodiment, a kitcomprising a first component having a ligand linked to a solid phaseadsorbing composition through a chemical spacer. The kit also includes asecond component having a ligand-binding moiety and the antigen orhapten that binds the antibody, and a third component comprising anantigen or hapten and a label. The kit can further include an antibodystandard.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a calibration curve for detection of high affinity anti-C6IgG antibodies added to normal human serum.

FIG. 2 shows that conjugates C6-StrAv, 2:1 and C6-BSA-HRP, 4:1:3 areless active in the detection of a high affinity antibody

FIG. 3 shows the concentration/OD curves for detection of low affinityanti-C6 antibodies containing both IgG and IgM using various conjugatesmixtures. The results shows that conjugates with one or less moleculesof C6 peptide per StrAv and HRP, which detects high affinity anti-C6antibodies with high sensitivity, fail to detect low affinityantibodies. Conjugates containing 2 or 4 molecules C6 peptide per onemolecule of StrAv/BSA detect antibody with relatively good sensitivity.Mixture of all four conjugates also have similar sensitivity indetection of low affinity antibody.

FIG. 4 shows the detection of antibodies in patient sample at an earlystage of Lyme disease. A combination of peptide conjugates with variousmolar ratios of peptide/StrAv/HRP provided good sensitivity.

FIG. 5 shows a comparison of sensitivity of antibody detection using aconventional IgG/IgM C6 ELISA test with the assay of current invention.Positive control serum from a C6 ELISA kit, containing patient sera withanti-C6 IgG was used as source of antibodies.

FIG. 6 shows the critical dependence of sensitivity for detection ofanti-C10 low affinity IgM antibodies from amount of peptide inconjugates.

FIG. 7 shows a calibration curve and assay for linearity of detection ofanti-C10 antibodies using affinity antibody from patient with earlystage of infection as a calibrator. The conjugates used were C10-StrAv2:1, 0.03 ug/ml, C10-BSA-HRP 4:1:3 0.08 ug/ml.

FIG. 8 shows the sensitivity for detection of anti-C6 and anti-C10antibodies in a serum sample at separate detection using mixtures ofconjugates for separate detection (C6-StrAv 0.5:1, C6-StrAv 2:1,C6-StrAv 2:1, C6-BSA-HRP 4:1:3) and (C10-StrAv 2:1, C10-BSA-HRP 4:1:3)and mixture containing all conjugates for detection both antibodies inone well.

FIG. 9 shows a comparison of two means for running the assay—i.e. withthe sample mixed with conjugates in a separate tube, incubated for 10min than transferred into wells with capture reagent and with the samplemixed with conjugates directly into wells with capture reagent.Conjugates were C6-StrAv 0.5:1, 0.03 ug/ml C6-HRP 1:1, 0.05 ug/ml.

FIG. 10 shows the dependence of antibody concentration on OD over a widerange of antibody concentrations (anti-C6 high affinity, anti-C6 lowaffinity and anti-C10 IgM). A high dose hook effect was found only forthe detection of high affinity anti-C6 IgG and observable (OD dropsbelow OVER in ELISA reader with upper limit 3.5) at high antibodyconcentration exceeding range of linear response almost 10 times. Lowaffinity anti-C6 IgG/IgM and anti-10 IgM does not show a hook effect,which can be recognized in assay conditions at concentration of up to100 ug/ml.

FIG. 11 shows the effect of titration of biotin-binding sites inC6-StrAv conjugates with Bi-PEG on assay sensitivity. Blocking of onebinding site dos not significantly change the analytical sensitivity.The presence of only one to four biotin-binding sites was sufficient tomaintain activity peptide-StrAv conjugates in the test.

FIG. 12 shows scatter plots of OD for a panel of normal blood donors(412 samples) tested in parallel in conventional IgG/IgM C6 ELISA (atdilution 1:20) with C6 peptide immobilized on solid phase and new teston undiluted sera as described in Example 12.

FIG. 13 shows histogram of OD distribution for panel of 314 samples ofLyme patients or patients with suspected Lyme disease tested in parallelin conventional IgG/IgM C6 ELISA test at dilution 1:20 and with new test(Example 12) on undiluted samples. The number of samples which have ahigh OD values including OVER were significantly higher for new testwhich demonstrates the high analytical sensitivity of a new test.

FIG. 14 shows the application of C6-conjugates in a competitive formatfor the detection of whole protein (rec VLSe) containing C6 peptide.Less than 1 ng whole VLSe in a sample can be detected by this method.

FIG. 15 illustrates a schematic diagram showing the molecularinteractions underlying the immunoassays of the present invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention provides, in one embodiment, an immunochemicalmethod and reagents for qualitative or quantitative detection ofanalytes in biological fluids, including detection of antibodies tosmall antigens, epitopes, peptides or haptens.

The present method, in one embodiment, can be based on the ability of anantibody to bind at least two molecules of the same antigenic substance,due to the presence of at least two specific, substantially identicalantigen-binding sites on the antibody. For instance, the IgG class hastwo identical binding sites, while the IgM class has up to 10 identicalbinding sites.

The principle of antibody detection in the present assay involvesformation in solution of a specific immune complex between the antibodyto be detected and two molecules of antigen/hapten bearing two differentlabels. As shown in FIG. 15, one molecule 150 of antigen/hapten may beconjugated with a detector label 151, while a second molecule 152 may beconjugated to a component 153 of a high-affinity pair (e.g.,biotin-streptavidin) that allows specific capture of the ternary immunecomplex 154 thus formed onto a solid phase 155 coated with the othercomponent 156 of the affinity pair. The two conjugates 150 and 152 maybe added as a mixture to the sample being analyzed. In principle, oneconjugate 150 can bind to one antigen-binding site of the antibodymolecule 157 to be detected, while the second conjugate 152 can bind tothe other antigen-binding site on the same antibody molecule 157.Ternary complex 154 thus formed is bound to the solid phase 155 via thespecific interaction of the second conjugate 152 and its associatedpartner 153 of the affinity pair with the other affinity partner 156immobilized on the solid phase 155. An appropriate detection method(enzymatic or other) can then be applied to detect binding of theantibody-antigen complex. A single washing step may be needed to removeexcess reagent prior to quantitation of bound detector label. Most knowntypes of labels (enzymatic, fluorescent, chemiluminescent, colloidalgold, other colloids, colored particles etc.) used in variousimmunoassays can be utilized in tests based on this principle.

Furthermore, the detection principle employ by the present invention maybe applicable to various test formats. For example, it can be shown thatthe same reagents used for creation of a microplate C6 ELISA canfunction effectively in membrane ELISA formats, including rapid tests(performed in 5–10 min), in which sensitivity equal to or higher thanthat of the microplate ELISA format was demonstrated. Moreover, thepresent method can also be useful in antibody detection using rapidimmunofiltration and immunochromatographic assay techniques whileexhibiting very low background and non-specific reactions. The method ofthe present invention may also be especially suitable for application inimmunochromatographic serological tests.

The present method, in accordance with an embodiment, can besubstantially free from most interfering factors related tocross-reactivity problems, can work effectively with undilutedserum/plasma samples, can provide extremely low signal for normalsamples, and has significantly higher analytical sensitivity. Diagnosticsensitivity and specificity may also be increased with the presentmethod.

Assay protocols for use with the present invention can be relativelysimple and more convenient, with short turnaround time. For instance, anentire test can be performed within 30 minutes. The need for sampledilution may also be eliminated, as undiluted sample and conjugates canbe mixed directly in wells of a coated plate, followed by a single washstep. As such, testing of multiple samples can be especially convenientbecause of the insensitivity of results to prolonged incubation betweenadditions of sample and conjugates.

In addition, a broad range of linearity of absorbance (OD) values,covering almost the full scale of a typical ELISA reader in optimizedassay conditions may be achieved with a method of the present invention,making such method valuable for quantitative determination ofanti-peptide antibody concentration. For instance, the detection limitfor IgG antibody using the method of the present invention with HRPlabel and commercially available tetramethylbenzidene (TMB)-basedchromogenic substrates can be as low as 5 ng/ml in a 30 min test. Thelow background and low non-specific reactivity makes the method of thepresent invention suitable for further application of various signalamplification techniques for antibody detection.

Furthermore, the method of the present invention provides, in oneembodiment, an assay that is not species-dependent. Specifically,detection of antibodies against a specific antigen can be applied,without special modifications, for detection of antibodies in human, aswell as animal sera/plasma. Moreover, the present method can, in anembodiment, detect antibody classes involved in the humoral immuneresponse (e.g., IgG, IgM, IgA, IgE), such that analysis of the antibodyclass involved in specific immune complex formation with the labeledantigen can be done as a second step using appropriate secondaryconjugates.

In general, the present method allows for increased sensitivity ofdetection of antibody classes that have more than two binding sites forantigens, for instance, IgM pentamer or IgA dimer. As such, the presentmethod can narrow the “window period” for serological detection ofinfection due to more sensitive detection of IgM antibodies during theearly phase of seroconversion. In addition, the present method can bevaluable for detection of secretory IgA antibodies in various secretionscontaining the first line of humoral antimicrobial defense (e.g.,saliva, intestinal, respiratory, erythema, gastrointestinal).

In another embodiment, the present method permits an application of newimmune complex-forming reagents for quantitative detection of an antigen(e.g., VlsE protein). Sensitivity of better than 1 ng/ml can be attainedusing a version of the new test in combination with anti-C6 peptideantibody.

In certain preferred embodiments, the present invention takes advantageof the ability of all natural antibodies to bind at least two identicalmolecules of antigens by binding one antigen or hapten though each ofthe two arms of the immunoglobulin structure. In preferred embodimentsthe invention utilizes two basic types of peptide antigen conjugateswhich are recruited into a specific immune complex by the antibody to bedetected. The resulting immune complex may be selectively captured on asolid phase without interference from even a large excess ofnon-specific immunoglobulins present in sample. The presence of adetector label on one of the two peptide antigen conjugates allows forthe detection and, in certain embodiments, the quantitation of thespecific immune complex, which is proportionate to the amount ofspecific peptide-binding antibody that is present in the test sample.

In preferred embodiments, the first peptide is conjugated directly orindirectly to a detector label by means known in the art so that thestructure of the epitope recognized by the antibody is retained (forexample, a selected functional group may be attached to one the terminalaminoacids). This conjugate is herein referred to as the third componentor the third conjugate component of the immunassay or of the presentinvention. The second peptide conjugate according to the presentinvention is the peptide conjugated directly or indirectly to partner ofbioaffinity pair (i.e. to a ligand-binding moiety or the ligand to whichit binds-preferably with a high affinity). In preferred embodiments thishigh affinity binding partner has a higher mol weight than itsfunctional (affinity) counterpart. This conjugate of the peptide to thecomponent of the bioaffinity pair is herein referred to as the secondcomponent or the second conjugate component of the immunassay of thepresent invention. These conjugates should preserve functional activityof both biologically active components—i.e. of both the antigenicantibody-binding activity of the peptide as well as the binding activityof the bioaffinity partner. The third component of the systemparticipating in specific antibody detection is a conjugate of thesecond component of the bioaffinity pair (i.e. a ligand where aligand-binding moiety is incorporated into the second component and aligand-binding moiety where a ligand is incorporated into the secondcomponent) with a macromolecular carrier which can be (or has been)immobilized on a solid phase. The invention teaches the preparation of amultiplicity of reagents of the capture system which provide efficientadsorption of reagent on various types of solid phases with high bindingcapacity for labeled specific immune complex. The effect of the presenceof components of the bioaffinity partners in human blood (e.g. samplescontaining immunoglobulins where the bioaffinity partners are antibodyand antigen) also has been estimated. Factors important for quantitativedeterminations of specific antibodies, high dose hook effect(prozoning), linearity of dependence antibody concentration-signal arealso provided by the invention.

The invention provides means for optimizing the efficiency of antibodydetection by determining the affinity of the antibody to be detected ormeasured by methods (as compared with conventional methods using solidphase immobilized peptides). In preferred embodiments, the inventionincludes methods that preferably include: optimization of peptideconjugates in respect of amount of peptides in each type of conjugates(as a molar ratio); optimization of the distribution of peptide betweentwo types of conjugates; and optimization of their final concentrationsin the final immunoassay. The efficacy of solid phase immunoassay isbased on high local concentrations of analytes (e.g. antibodies andantigens) associated with adsorption on solid phase—which promotes theimmunological antigen-antibody reaction through the law of mass action.Special approaches are known in the art for preparation of peptideantigens and their adsorption on a solid phase which increaseconcentration of peptides on solid phase (polymers with multiplepeptides, peptides with repeating epitopic sequence ets). Frequentlyused approaches for peptide immobilization are based on the use ofStreptavidin (StrAv) and biotinylated peptide, which theoreticallypermit for formation of immobilized complexes containing up to fourpeptides in a single immobilized complex. TheBiotin/Streptavidin/Avidin/Antibiotin bioaffinity systems currentlyavailable are provide the most versatile system with variousapplications in area of bioanalytical and diagnostic detection system.The high affinity and specificity of biotin-streptavidin interaction,stability of StrAv, presence of four biotin-binding sites, commercialavailability of reagents contribute to making this system the preferredone among bioaffinity pairs available. This system was utilized in themethod of the instant invention as a preferred system for demonstratingthe advantages of the invention. Typically, StrAv is used as solid-phaseimmobilized reagent as component for immobilization of biotin-labeledspecific ligands or additional components of detection systemparticipating in specific capture of analytes, while biotin is typicallyused for the labeling of specific or supplementary analytes and detectorlabels. In contrast, the present invention demonstrates the advantagesof reversing these roles of StrAv and biotin counterparts.

The development of a test system for detection of antibodies against twopeptide antigens is important for, for example, serological diagnosis ofLyme disease. The invention provides methods for the detection ofdifferent types of immune responses. For example, one antigen peptidecharacteristic of Lyme disease is peptide C6, representing aconservative region of one of the variable domains of VLSe of B.burgdorferi. C6 is known as an epitope which elicit primarily IgGresponse, typical seroconversion from IgM to IgG response has not beenfound for this peptide, although IgM components can be detected (see,e.g., Liang F. T at al. (1999) J. Clin. Microbiol. 37:3990–3996). Thesecond peptide, C10 representing an immunogenic C-terminal epitope ofOspC protein of B. burgdorferi is known as an epitope which results in apersistent IgM response at all stages of disease without typical IgM-IgGswitching. The C10 peptide is considered to be antigen which, ifdetected, can improve the sensitivity and accuracy of diagnosis of earlyLyme borreliosis (see Mathiesen M. J. et al. (1998) J. Clin. Microbiol.36:3474–3479).

Both peptides C6 and C10 modified at N-terminal with biotin andimmobilized on solid phase through StrAv bridge work well as antigens inconventional assays using anti-human IgG/IgM enzyme conjugates. Covalentconjugates of both peptides through N-terminal Cys with maleimideactivated BSA as described in Examples also function well as reagentsfor immobilization on solid phase. Cross-reactive results have beenfound, however, for some sera samples related to the presence ofantigenic substances on the solid phase not related to the specificpeptide epitopes. The method of the invention provides an additionaltest for analysis of specificity of the immunoassay signal based oninhibition of signal using addition of an excess of non-modified purepeptide to diluted sera samples before transferring into antigen coatedwells. Applied to the Lyme antigens, this approach has shown that C6peptide can inhibit very efficiently specific signals at very lowconcentration, but C10 can not. This phenomenon was examined through themethod of the invention by analysis of inhibitory activity of variouspeptide-BSA and peptide-StrAv conjugates.

In general the method of the invention provides an immunoassay whichprovides unique compositions, but also utilizes components andmethodologies used in the art. For example, U.S. Pat. Nos. 4,778,751,4,945,042, 5,236,849, 5,312,730, 5,705,338, 5,989,806, 6,030,770 and6,121,006, the contents of which are incorporated by reference herein intheir entirety, provide standard methodologies and reagents for use inthe method of the present invention.

As used herein, a “label” is any molecule, which produces or can beinduced to produce a signal. The label may be conjugated to an analyteor an antibody, or to another molecule such as a receptor or a moleculethat can bind to a receptor such as a ligand, particularly a hapten. Inthe subject invention, the label can be a member of the signal producingsystem, as defined below, that includes a signal producing means.

The label may be isotopic or nonisotopic, preferably nonisotopic. By wayof example and not limitation, the label can be a part of a catalyticreaction system such as enzymes, enzyme fragments, enzyme substrates,enzyme inhibitors, coenzymes, or catalysts; part of a chromogen systemsuch as fluorophores, dyes, chemiluminescers, luminescers, orsensitizers; a dispersible particle that can be non-magnetic ormagnetic, a solid support, a liposome, a ligand, a receptor, a haptenradioactive isotope, and so forth.

Enzymes, enzyme fragments, enzyme inhibitors, enzyme substrates, andother components of enzyme reaction systems can be used as labels. Whereany of these components is used as a label, a chemical reactioninvolving one of the components is part of the signal producing system.

When enzymes are employed, molecular weights of the label typicallyrange from about 10,000 to 600,000, more usually from about 10,000 to300,000, and the involved reactions will be, for the most part,hydrolysis or redox reactions.

Coupled catalysts can also involve an enzyme with a non-enzymaticcatalyst. The enzyme can produce a reactant, which undergoes a reactioncatalyzed by the non-enzymatic catalyst or the non-enzymatic catalystmay produce a substrate (includes coenzymes) for the enzyme. A widevariety of non-enzymatic catalysts, which may be employed are found inU.S. Pat. No. 4,160,645 (1979), the appropriate portions of which areincorporated herein by reference.

The enzyme or coenzyme employed provides the desired amplification byproducing a product, which absorbs light, e.g., a dye, or emits lightupon irradiation, e.g., a fluorescer. Alternatively, the catalyticreaction can lead to direct light emission, e.g., chemiluminescence. Alarge number of enzymes and coenzymes for providing such products areindicated in U.S. Pat. No. 4,275,149, columns 19 to 23, and U.S. Pat.No. 4,318,980, columns 10 to 14, which disclosures are incorporatedherein by reference.

A number of enzyme combinations are set forth in U.S. Pat. No.4,275,149, columns 23 to 28, which combinations can find use in thesubject invention. This disclosure is incorporated herein by reference.

Of particular interest are enzymes, which involve the production ofhydrogen peroxide and the use of the hydrogen peroxide to oxidize a dyeprecursor to a dye. Particular combinations include saccharide oxidases,e.g., glucose and galactose oxidase, or heterocyclic oxidases, such asuricase and xanthine oxidase, coupled with an enzyme which employs thehydrogen peroxide to oxidize a dye precursor, that is, a peroxidase suchas horse radish peroxidase, lactoperoxidase, or microperoxidase.Additional enzyme combinations may be found in the subject matterincorporated by reference.

When a single enzyme is used as a label, such enzymes that may find useare hydrolases, transferases, lyases, isomerases, ligases or synthetasesand oxidoreductases, preferably, hydrolases. Alternatively, luciferasesmay be used such as firefly luciferase and bacterial luciferase.Primarily, the enzymes of choice, based on the I.U.B. classificationare: Class 1. oxidoreductases and Class 3. Hydrolases; particularly inClass 1, the enzymes of interest are dehydrogenases of Class 1.1, moreparticularly 1.1.1, 1.1.3, and 1.1.99 and peroxidases, in Class 1.11. Ofthe hydrolases, particularly Class 3.1, more particularly 3.1.3 andClass 3.2, more particularly 3.2.1.

Illustrative dehydrogenases include malate dehydrogenase,glucose-6-phosphate dehydrogenase, and lactate dehydrogenase. Of theoxidases, glucose oxidase is exemplary. Of the peroxidases, horse radishperoxidase is illustrative. Of the hydrolases, alkaline phosphatase,beta-glucosidase and lysozyme are illustrative.

Those enzymes, which employ nicotinamide adenine dinucleotide (NAD) orits phosphate (NADP) as a cofactor, particularly the former, can beused. One preferred enzyme is glucose-6-phosphate dehydrogenase,preferably, NAD-dependent glucose-6-phosphate dehydrogenase.

The label can also be fluorescent either directly or by virtue offluorescent compounds or fluorescers bound to a particle or othermolecule in conventional ways. The fluorescent labels will be bound to,or functionalized to render them capable of binding (being conjugated)to, optionally through a linking group, cyclosporin or antibodies orreceptors for cyclosporin.

The fluorescers of interest will generally emit light at a wavelengthabove about 350 nm, usually above about 400 nm and preferably aboveabout 450 nm. Desirably, the fluorescers have a high quantum efficiency,a large Stokes shift, and are chemically stable under the conditions oftheir conjugation and use. The term luminescent label is intended toinclude substances that emit light upon activation by electromagneticradiation, electro chemical excitation, or chemical activation andincludes fluorescent and phosphorescent substances, scintillators, andchemiluminescent substances.

Fluorescers of interest fall into a variety of categories having certainprimary functionalities. These primary functionalities include 1- and2-aminonaphthalene, p,p-diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminostilbenes imines,anthracenes, oxacarboxyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazine, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene,7-hydroxycoumarin, 4,5-benzimidazoles, phenoxazine, salicylate,strophanthidin, porphyrins, triarylmethanes, flavin and rare earthchelates, oxides, and salts. Exemplary fluorescers are enumerated inU.S. Pat. No. 4,318,707, columns 7 and 8, the disclosure of which isincorporated herein by reference.

Energy absorbers or quenchers can be employed either separately or inconjunction with one another. The absorber or quencher can additionallybe bound to a solid insoluble particle of at least about 50 nm indiameter. When the distance between the absorber and the quencherresulting from specific binding events (such as antibody-antigenbinding) too small, the fluorescence of the absorber is quenched by thequencher. The quencher may be the same or different, usually different,from the fluorescer.

An alternative source of light as a detectable signal is achemiluminescent source, and, therefore, a label can be achemiluminescent compound. The chemiluminescent source involves acompound, which becomes electronically excited by a chemical reactionand may then emit light which serves as the detectable signal or donatesenergy to a fluorescent acceptor.

A diverse number of families of compounds have been found to providechemiluminescence under a variety of conditions. One family of compoundsis 2,3-dihydro-1,4-phthalazinedione. The most popular compound isluminol, which is the 5-amino analog of the above compound. Othermembers of the family include the 5-amino-6,7,8-trimethoxy- and thedimethylamine-[ca]benzo analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino- and para-methoxy-substituents.Chemiluminescence may also be obtained with geridinium esters,dioxetanes, and oxalates, usually oxalyl active esters, e.g.,p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basicconditions. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins.

Conjugate—A conjugate is a molecule comprised of two or more subunitsbound together, optionally through a linking group, to form a singlestructure. The binding can be made either by a direct connection (e.g. achemical bond) between the subunits or by use of a linking group. Forexample, in one context of the present invention, a peptide antigen isconjugated, optionally through a linking group, a detectable label. In asecond context, the peptide antigen is conjugated to a member of ahigh-affinity binding pair (i.e. preferably a ligand-binding moiety).

Conjugation—Conjugation is any process wherein two subunits are linkedtogether to form a conjugate. The conjugation process can be comprisedof any number of steps.

Receptor—A receptor is any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule.These organized areas of a molecule are referred to as epitopic ordeterminant sites. Illustrative naturally occurring receptors includeantibodies, enzymes, fab fragments, poly(nucleic acids), complementcomponent, i.e. thyroxine binding globulin, lectins, protein A, and thelike. Receptors are also referred to as antiligands. A natural receptorexists that binds specifically to cyclosporin.

Ligand—A ligand is any organic molecule for which a receptor naturallyexists or can be prepared. For example biotin is a high-affinity ligandwhich binds to avidin (or strepavidin).

Hapten—Haptens are capable of binding specifically to correspondingantibodies, but do not themselves act as immunogens (or antigens) forpreparation of the antibodies. Antibodies which recognize a hapten canbe prepared against compounds comprised of the hapten linked to animmunogenic (or antigenic) carrier. Haptens are a subset of ligands.

Member of a specific binding pair—A member of a specific binding pair(sbp member) is one of two different molecules, having an area on thesurface or in a cavity, which specifically binds to and is therebydefined as complementary with a particular spatial and polarorganization of the other molecule. The members of the specific bindingpair are referred to as ligand and receptor (antiligand). For example,avidin and biotin form a preferred specific binding pair. The specificbinding pair may also be members of an immunological pair such asantigen-antibody, although other specific binding pairs, such asbiotin-avidin, hormones-hormone receptors, nucleic acid duplexes,IgG-protein A, DNA-DNA, DNA-RNA, and the like, are not immunologicalpairs but are specific binding pairs.

A solid phase (or support or surface)—A solid phase is a porous ornon-porous water insoluble material. The solid phase can be hydrophilicor capable of being rendered hydrophilic and includes inorganic powderssuch as silica, magnesium sulfate, and alumina; natural polymericmaterials, particularly cellulosic materials and materials derived fromcellulose, such as fiber containing papers, e.g., filter paper,chromatographic paper, etc.; synthetic or modified naturally occurringpolymers, such as nitrocellulose, cellulose acetate, poly(vinylchloride), polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), etc.; either used by themselves or in conjunction with othermaterials; glass available as Bioglass, ceramics, metals, and the like.Natural or synthetic assemblies such as liposomes, phospholipidvesicles, and cells can also be employed. Other materials, which can beemployed, are described above in the definition of immunogenic carrierparticles and below in the definition of a signal producing system.Other preferred solid phase membrane materials are described below inthe examples.

The binding of members to the support or surface may be accomplished bywell-known techniques, commonly available in the literature, anddescribed above in the definition of immunogenic carrier particles. Thesurface can have any one of a number of shapes, such as strip, rod,particle, including bead, and the like. The surface will usually bepolyfunctional or be capable of being polyfunctionalized or be capableof binding an sbp member through specific or non-specific covalent ornon-covalent interactions. A wide variety of functional groups forlinking are described in the definition of immunogenic carrierparticles.

The length of a linking group or chemical linker” to the peptide antigenor ligand may vary widely, depending upon the nature of the compoundbeing linked, the effect of the distance between the compound beinglinked and the support on the assay and the like.

Signal producing system—The function of the signal producing system isto produce a product, which provides a detectable signal related to theamount of bound and/or unbound label. The signal producing system mayhave one or more components, at least one component being a label. Thesignal producing system includes all of the reagents required to producea measurable signal including signal producing means capable ofinteracting with the label to produce a signal. The signal producingsystem provides a signal detectable by external means, normally bymeasurement of electromagnetic radiation, desirably by visualexamination. For the most part, the signal producing system includes achromophoric substrate and enzyme, where chromophoric substrates areenzymatically converted to dyes, which absorb light in the ultravioletor visible region, phosphors, or fluorescers.

The signal producing means is capable of interacting with the label toproduce a detectable signal. Such means include, for example,electromagnetic radiation, heat, chemical reagents, and the like. Wherechemical reagents are employed, some of the chemical reagents can beincluded as part of a developer solution. The chemical reagents caninclude substrates, coenzymes, enhancers, second enzymes, activators,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Some of the chemical reagents such as coenzymes, substances thatreact with enzymic products, other enzymes and catalysts, and the likecan be bound to other molecules or to a support.

The signal producing system including the label can include one or moreparticles, which are insoluble particles of at least about 50 nm and notmore than about 50 microns, usually at least about 100 nm and less thanabout 25 microns, preferably from about 0.2 to 5 microns, diameter. Theparticle may be organic or inorganic, porous or non-porous, preferablyof a density approximating water, generally from about 0.7 to about 1.5g/mL, and composed of material that can be transparent, partiallytransparent, or opaque. Generally, particles utilized as a label willhave similar characteristics to those described above in the definitionsof an immunogenic carrier and a support or surface.

Many different types of particles may be employed for modulating lightemission. Of particular interest are carbon particles, such as charcoal,lamp black, graphite, colloidal carbon and the like. Besides carbonparticles metal sols may also find use, particularly of the noblemetals, gold, silver, and platinum. Other metal-derived particles mayinclude metal sulfides, such as lead, silver, or copper sulfides ormetal oxides, such as iron or copper oxide.

Fluoresceinated latex particles are taught in U.S. Pat. No. 3,853,987and are available commercially as Covaspheres from Covalent TechnologyCorp.

Quantitative, semiquantitative, and qualitative methods as well as allother methods for determining antibody or antigen are considered to bemethods of detecting the antibody or antigen. For example, a method thatmerely detects the presence or absence of an antibody in a samplesuspected of containing the antibody is considered to be included withinthe scope of the present invention.

Synonyms for the phrase “measuring the amount of antibody”, which arecontemplated within the scope of the present invention include, but arenot limited to, detecting, measuring, or determining antibody;detecting, measuring, or determining the presence of antibody; anddetecting, or determining the amount of antibody. The same principleapplies to the phrase “measuring the amount of antigen”.

An “antibody sample” is a sample suspected of containing anantibody—i.e. any sample, which is reasonably suspected of containing anantibody, can be analyzed by the method of the present invention. Suchsamples can include human, animal, or man-made samples. The sample canbe prepared in any convenient medium, which does not interfere with theassay. Typically, the sample is an aqueous solution or a natural fluid,preferably, urine, whole blood, serum, plasma, or saliva morepreferably, whole blood.

In the methods of the invention the antibody to be detected is aspecific immunoglobulin, preferably a specific IgA, IgD, IgE, IgG, IgM,and subclasses thereof, and more preferably a specific IgE, or a classof antibodies, such as immunoglobulins, preferably selected from thegroup consisting of total IgA, total Igd, total IgE, total IgG, totalIgM and isotypes thereof, most prefer ably total IgE.

The ligand antigen, antibody or hapten bound to biotin can be anyimmunologically active substance, such as an allergen, antibodies, suchas polyclonal antibodies, monoclonal antibodies including recombinantantibodies or fragmented antibodies, preferably an allergen and/or apolyclonal anti-immunoglobulin, such as goat anti-human polyclonal serumsupplied by Ventrex Laboratories, Inc., Portland, Me., Catalog No.77660. In the reference immuno-assay said antibody is preferablydirected against the constant portion of the class of antibodies to bemeasured, i.e. an antibody directed against the IgE-antibodies.

By biological fluid is meant any clinical sample, such as blood, plasma,serum, urine or saliva, which also includes any biological fluid whichis excreted, secreted or transported internally in an organism.

EXAMPLE

The following detailed guidance and examples are provided to furtherillustrate and define preferred aspects of the invention.

Detecting Anti-Lyme Disease Antibodies

Affinity-purified anti-peptide antibodies were isolated (Example 6) fromtwo different patient sera pools—one from patients with late Lymedisease, which contained only IgG antibodies against the C6 peptide, andthe second from patients at early stage of Lyme disease containing bothIgG and IgM classes of antibodies against C6 and C10 peptides. Affinitypurification was conducted according to conventional ELISA test withsolid-phase immobilized peptides (see Example 13). A series of C6-BSA,C10-BSA, C6-StrAv and C10-StrAv conjugates at various ratios ofpeptide/BSA (from 1:1 to 8:1) have been synthesized and tested forinhibitory activity for IgG and IgM antibodies in preparations ofaffinity purified anti C6 and anti-C10 antibodies (see Examples 7 and14). Table 1 below illustrates the differences in inhibitory activity(which reflects affinity) seen with non-conjugated peptides and withpeptides having various molar ratios of peptide/BSA as well as thesource of the antibody—i.e. early or late stage of disease.

TABLE 1 Concentrations of non-conjugated peptides and peptide-BSAconjugates which induce 50% inhibition of binding of anti-peptideantibodies with solid-phase immobilized peptides Anti-C6 IgG Anti-C6 IgGAnti-C6 IgM High affinity Low affinity Low affinity C6 non-conjugated1–1.5 ng/ml 10–15 ng/ml 5–8 ug/ml C6-BSA 1:1 0.6–0.8 ng/ml 5–10 ng/ml10–20 ng/ml C6-BSA 2:1 0.6–0.8 ng/ml 1.5–2.0 ng/ml 5–10 ng/ml C6-BSA 4:10.6–0.8 ng/ml 0.8–1.0 ng/ml 2–4 ng/ml C6-BSA 8:1 0.6–0.8 ng/ml 0.4–0.6ng/ml 1–2 ng/ml Anti-C10 IgG Anti-C10 IgM Low affinity Low affinity C10non-conjugated 50–80 ug/ml 80–100 ug/ml C10-BSA 1:1 3–4 ug/ml 3–4 ug/mlC10-BSA 2:1 1.5–2.0 ug/ml 1–2 ug/ml C10-BSA 4:1 0.6–1.0 ug/ml 1–2 ug/mlC10-BSA 8:1 0.1–0.2 ug/ml 30–60 ng/ml

The binding of anti-C6 IgG antibodies isolated from late serum withsolid-phase immobilized peptide inhibited with almost equal efficiencyto non-conjugated C6 peptide and all C6-BSA conjugates. For anti-C6antibodies isolated from serum of patient at early stage of Lyme diseaseinhibitory activity (affinity) of non-conjugated C6 peptide and C6-BSAconjugates differ significantly. There was a direct relationship betweenthe amount of peptide in BSA conjugates and its activity as aninhibitors of IgG and IgM anti-C6 antibody activity in solution. Evenmore significant is the difference between non-conjugated peptide andBSA-C10 conjugates in inhibition of IgG and IgM anti-C10 antibodies.Almost three order of magnitude difference between the concentration ofnon-conjugated C10 and C10-BSA 8:1 conjugate was required to reduce thebinding of anti-C10 antibodies by 50% in solution with solid-phaseimmobilized peptide. These results demonstrate that, optimally, morethan one peptide should be present in peptide conjugates to optimizebinding in solution with specific anti-peptide antibodies having lowaffinity to peptide epitopes.

In contrast, for the formation of complexes with high affinityantibodies one peptide in the peptide conjugate may be enough (FIG. 1).Conjugates of C6 and C10 peptides with detector label (HRP) wereprepared two ways. First, conjugates containing 1 or less molecules ofpeptide per HRP molecule were prepared by direct reaction ofCys-peptides with maleimide activated HRP (see Examples 7 and 8). HRPdoes not have enough amino groups to introduce more maleimide groupsconveniently. Conjugates of peptide-HRP with more than one peptide wereprepared by conjugation of peptide-BSA conjugates having reactivemaleimide groups with thyolated HRP, prepared as described in Example 4.The presence of approximately one SH groups in HRP in this case favorsformation of conjugates without cross-linking problem. Conjugatescontaining up to 4.5 HRP molecules per one peptide_BSA-maleimide wereprepared (Example 8).

As a major component of solid phase capture system the current inventiondescribes biotinylated BSA where biotin is attached to BSA through a PEGspacer with MW3500 (Example 5). Biotechnical and biomedical applicationof PEG is known in the art [see, e.g., Poly(ethylene glycol) chemistryEd by J. M. Harris, Plenum Press, New York, 1992]. The characteristicsof PEG which favor its use as a spacer or linker for use in the presentinvention include its: hydrophilic characteristics, absence of charge,flexibility, stabilizing effect on proteins, and reduction ofimmunogenic properties of proteins. Accordingly, Bi-PEG-BSA is versatilereagent for adsorption on various solid phases, polystirol plates, NCand other membranes in this invention (in which high binding capacity tobiotin-binding proteins and reduced non-specific adsorption effect areoptimal). Bi-PEG-BSA provides enhanced affinity to the solid phase,stable adsorption, and can be applied at a concentration which may beenough for simultaneously efficient blocking of the solid phase, therebyeliminating requirement for secondary blocking reagents. As a result,the procedure for preparation of coated devices become very simple andinexpensive.

In one of embodiments of this invention the application of PEG-modifiedstreptavidin is described in the examples which follow. The PEG-modifiedstreptavidin may be used to enhance adsorption on various solid phaseswith reduced NSB reactions. StrAv, modified through amino groups withPNC activated StrAv MW 5000 has enhanced and stable adsorption onpolystirol plates and NC membranes, non-specific adsorption ofimmunoglobulins from sera samples was also reduced and potentialimmunogenic epitopes on streptavidin also appear to be sheltered.Another application of PEG-modified StrAv, where effect of modificationis very pronounced is adsorption on colloidal gold particles. PEG-StrAvcontrary to non-modified StrAv adsorbs on colloidal particles at neutralpH at lower protecting concentration with formation stable and moreactive conjugates than colloidal gold conjugates prepared by adsorptionof native streptavidin at optimal pH.

An important advantage of a method with dual labeled antigen isreduction of the nonspecific reactions and some cross-reactivereactions, which can contribute to false—positive results inconventional tests. The application of new peptides conjugates foranalysis of a big population of normal sera samples have shown that ODvalues in new test are significantly lower than for the same populationtested in conventional ELISA tests. For most sera tested as undilutedsamples OD values were equal to the blank reagent. Low cutoff values canbe calculated for new tests using various approaches, demonstrating thatdual labeled antigen method better discriminate positive and negativeresults (see FIG. 12)

The formation of triple complex of anti-peptide antibody with twodifferent peptide conjugates depends on several factors includingantibody affinity, antibody class, concentration and the amount ofpeptide in the conjugates as well as the molar ratio of peptides andantibodies in the sample. To initiate specific complex formation insolution the two conjugates sample should be mixed to obtain a solutioncontaining both conjugates. Several reaction products will be formedincluding: first, a complex of antibodies with peptide-detector label(complex 1), second, a complex of antibody with peptide conjugates ofthe second component of the bioaffinity pair (complex 2), and, finally,a triple complex of antibody with the two different peptide conjugates(complex 3). Complex 1 contains label, but can not be captured on solidphase, while complex 2 can be captured but does not contains label andcan not detected after washing. Only triple complex 3 can be capturedand detected. Antibody with only two antigen-combining sites (IgG, IgE)can be captured and detected only in complex with equal amount of bothconjugates. Complexes of antibodies having four or moreantigen-combining sites can be captured and detected at any ratio ofconjugates in complex allowed by antibody structure at the conditionthat at least one of each should be present in the complex. For,example, antibodies of the IgM class containing up to 10antigen-combining sites conditions which allow for complex formationwith one molecule of peptide conjugate needed for capture and several ofthe peptide conjugates with label will provide for higher sensitivity ofantibody detection because more label will present per each antibody incaptured complex (see FIG. 6). This is only simple schematic picture.

Other combinations of dually-labeled antigens with antibodies are alsopossible-especially for conjugates containing more than one antigenmolecule, when more than one antibody can be involved in complex withone polyepitopic (multiantigenic) conjugate. According to this schemethe likelihood (statistical probability) of formation of a triple immunecomplex is higher when total antigen concentration (amount) exceed theamount of specific antibodies—i.e. the method of the invention isparticularly well suited for antibody detection in low titer samples.When antibody concentration in the antibody sample increases at aconstant concentration of antigen in the conjugates, the relative ratioof complex of one type of conjugate/triple immune complex (portion ofcomplexes with only one conjugates) increases—which can lead atappropriate ratios to formation of small amounts of triple complex andas a result to a decrease in the signal for samples with highconcentration of antibodies (high dose hook effect). Using affinitypurified anti-peptide antibodies spiked into normal human serum, it wasfound that high dose hook effect can be observed for high affinityantibodies at concentrations which significantly exceed concentration inthe linear range for OD/concentration (see FIG. 10). This effect can notlead to false negative results, because it is observed at concentrationsrarely present in patient samples and signals do not drops to very lowlevels. The sensitivity of antibody detection depends on the ratiobetween capture capacity of the solid phase in respect to thebioaffinity partner and the concentration of conjugate of antigen withthe affinity counterpart. If the binding capacity of the solid phasewith immobilized affinity partner is significantly less than the amountof conjugated counterpart in the sample, then reduction of sensitivitymay arise as result of competition between conjugates not involved inimmune complex formation and involved in triple or dual immune complex.Decreasing the conjugate concentration can minimize this effect, but atthe same time sensitivity may drop as a result of the lowering ofconcentration of reagents with a concomitant reduction of antibodyconcentration range which can tolerate the high dose hook effect.Accordingly, by these considerations, a solid phase which can exceptlarge amounts of capture reagent, exceeding the amount of reagents insolution and not a limiting factor for selection of conjugateconcentration, can provide for the highest levels of sensitivity.

Various membrane formats including dot-blot, flow-through and lateralflow formats which use membranes with high binding capacity arepotentially good formats for application of the method of the currentinvention. Several membrane formats were developed for antibodydetection using Bi-PEG-BSA as a capture reagent and peptide-StrAv,peptide-HRP and peptide-colloidal gold conjugates to demonstrate theadvantages of dual labeled antigen method (see Examples 10, 11, 15, 16,17, 18). Both membrane tests using HRP as a detector label, dot-blot(Example 17) and flow-through (Example 15) have shown analyticalsensitivity exceeding the sensitivity of conventional IgG/IgM ELISA(Example 13) according to a comparison of detection limits for seriallydiluted positive sera. Membrane versions with colloidal gold conjugates(see Examples 16 and 17) also work well and clearly discriminatenegative and positive sera. Colloidal gold conjugates of C6 peptide withC6-StrAv were also applied in lateral flow format as dry reagents inconjugate pad and Bi-PEG-BSA striped on membrane as capture line. Cleardiscrimination between negative and positive sera have been found.

The method of the current invention accepts that formation of the immunecomplex with dual-labeled antigens and its capture can proceed as asingle step when samples containing antibody are mixed directly withconjugates in coated solid phase. This then requires a single washingstep after incubation. The process of specific complex formation and itscapture can also be carried out in two steps. In this case, the sampleis first incubated with conjugates and subsequently transferred intosolid phase with capture reagent (see FIG. 9). Simultaneous incubationof the sample with peptide conjugates and a single washing step wasespecially advantageous in various tests using plates, tubes, and ballsas a solid phase. Significant simplification and shortening of assayprocedures for antibody detection can be achieved. As a furtheroptimization of assay procedure, agitation may be used for accelerationof a capture process that further shortens the assay time and improveslinearity of the dependence of signal/antibody concentrations. Inmembrane based tests, preincubation of sample with conjugates can be theprocedure of choice. In rapid lateral flow tests, conjugates can be usedas one reagent dried on one conjugate pad material or two antigenconjugates can be distributed among two separate conjugate pads. Theformation of a specific immune complex in this last cases will precedeby binding on capture line or membrane area with the applied capturereagent.

One of the embodiments of the current invention utilizes peptide(antigen) conjugates containing more than one peptide or polypeptide(antigen type) in the conjugate with detector label and bioaffinitypartner. Conjugates containing various antigens can be also mixed andused as single reagent for detection of several antibodies in a singlesample (see FIG. 8). When necessary, multiple antibody classes involvedin specific immune complex can be analyzed by adding a step ofincubation with class-specific antibody into solid phase containingcaptured immune complex with dual labeled antigens as described inExample 20. Repeating this with detection antibodies specific to all thevarious antibody classes provides a detailed picture of the immunestatus of antibody production in a patient antibody sample.

Reagents and assay principles developed in current invention can also beapplied for the detection of components of infectious agents bearingepitopes used for antibody detection i.e. for antigen detection. In thiscase (Example 19), sample, containing the antigen is first incubatedwith a small amount of epitope-specific antibody (this may be affinitypurified using anti-peptide antibody or whole sera containingpeptide-specific antibody), then the mixture is contacted with the solidphase containing the capture reagent and peptide conjugate componentsdescribed above. The epitope-bearing substance in the sample competeswith the peptide conjugates for antibody binding and reduces the signalin concentration dependent manner. Standard curves may be generatedusing known quantities of the epitope bearing substance (i.e. peptide orpolypeptide or protein bearing the antigen). FIG. 14 shows examples ofdetection of whole recombinant VLSe protein using this approach—whichdemonstrates that ng amount of antigenic substance can be readilydetected.

The following Examples and accompanying drawings are given for thepurpose of illustrating the present invention.

C6 and C10 Peptides, Sequences and Synthesis Procedure

Peptides C6 (26 AA) and C10 (12AA) were synthesized by automatic solidphase synthesis with the fluor-etylmetoxycarbonyl (Fmoc) strategy,followed by purification by HPLC and sequence verification by massspectroscopy. Purity of peptides was >90%. N-terminal Cys was includedinto both peptides and biotin also was included into N-terminal Cys ofC10 peptide during synthesis. C10 peptide also contains serine residuebetween N-terminal Cys and a sequence of ten aminoacids corresponding toC-terminal fragment of OspC. Biotinylated C6 peptide was prepared frompure Cys-C6 by modification with Biotin-PEG-Maleimid MW 3500 (ShearwaterPolymers, Inc) at equimolar ratio Cys and Maleimide in 0.1M sodiumphosphate buffer, pH 7.0. The structure and immunogenic properties ofboth peptides are well known in the art.

Example 1 Procedure for Detection of SH Groups in Peptides and ThiolatedProteins

The presence of SH groups in peptides, thyolated proteins and productsof conjugate synthesis through maleimide-sulfhydril cross-linking, andalso the immobilization of peptides on gels were analyzed using knownDTNB method in Tris buffer, pH 8.0, using molar absorbanceE_(412 TNB2-)=1.37×10⁴ cm⁻¹ M⁻¹.

Example 2 Conversion of Amino Groups of BSA, StrAv and HRP intoMaleimide Groups

Maleimide groups were introduced into BSA (Sigma), StrAv (Scipps Lab)and HRP (Scripps Lab) using GMBS(N-(gamma-Maleimidobutiryloxy)succinimide N-Succinimidyl4-maleimidobutyrate) in 0.1 M sodium phosphate buffer, pH 7.5,containing 1 mM EDTA at molar ratio maleimide/protein of 20:1 to 40:1with dialysis against the same buffer with pH 6.5 as a purificationstep. For quantitation of the number of maleimide groups, themaleimide-modified proteins were incubated with excess of DTT and afterexhaustive dialysis analyzed for presence of thiols in DTNB reaction.

The average number of maleimide groups introduced per each proteinmolecule was as follows: BSA-11, StrAv-7, HRP-1.1 (moles/mol protein).Protein concentration was determined spectrophotometrically usingadsorption coefficients E_(280 nm, 1 cm, 1 mg/ml)=0.66 and 3.0 for BSAand StrAv, respectively, and E_(403 nm, 1 cm, 1 mg/ml)=2.26 for HRP.

Example 3 Blocking of SH Groups in Peptides with Maleimide

To protect Cys-peptides from oxidation and dimer formation, freshsolutions of peptides were modified with N-ethylmaleimide in 0.1M sodiumphosphate, pH 6.5, with two-molar excess of maleimide.Maleimide-protected C6 and C10 were used in competition experiments asnon-conjugated peptide preparations.

Example 4 Preparation of HRP with Protected Sulfhydryls and Release ofLatent SH Groups

Latent SH groups were introduced into HRP using N-SuccinimidylS-acetylthiolacton (SATA) at 20–30 molar excess over HRP. Thiol groupswere released using treatment with hydroxylamine hydrochloride (20 mM)with subsequent dialysis overnight against 0.1M sodium phosphate, 1 mMEDTA, pH 6.5. Thiolated HRP after analysis of SH group content was usedimmediately for conjugation. The average number of SH groups was 1.2moles/mol HRP.

Example 5 Modification of BSA with Biotin and Biotin-PEG

BSA was biotinylated with Biotinamidohexanoic acidN-hydroxysuccinimidide ester at molar ratio biotin/BSA of 40:1 and withBiotin-PEG-N-hydroxisuccinimide at weight ratio BSA/Biotin-PEG-NHS of2:1 at BSA concentration 50–100 mg/ml in 0.1 M sodium phosphate, pH 8.1.Biotinylated BSA preparations were purified by dialysis againstPBS-sodium azide using dialysis membrane with cut-off 25,000 D.

Example 6 Preparation of C6-peptide and C10 Peptide Affinity Sorbentsand Procedure for Affinity Antibody Purification

Affinity sorbents containing C6 and C10 peptides were synthesized bycoupling Cys-peptides with thiol-specific Ultralink iodoacetyl gel(Pierce) in accordance with manufacturer's recommendations. Liganddensity was 0.24 mg/ml gel for C6 peptide and 0.4 mg/ml for C10 peptide.

Anti-peptide affinity antibodies were purified by passing high titerhuman serum (40–50 ml) from patients with Lyme disease, diluted twotimes with PBS-sodium azide buffer, through a column containing 3–4 mlof affinity gel at flow rate of 30 ml/hour. Columns were washed withPBS-sodium azide and bound anti-peptide antibodies eluted with Gentle™Ag/Ab elution buffer (Pierce). Elution buffer was removed by dialysisagainst PBS-sodium azide or desalting on Sephadex G-25 column.Concentration of affinity antibodies was estimated spectrofotometricallyusing A₂₈₀=1.4 and 1.18 of 1 mg/ml solution of IgG and IgM,respectively. The yield of antibodies was 4–6 mg from 40–50 ml serum.Purity and integrity of antibody preparations was confirmed by SDS-PAGEanalysis and sandwich ELISA test for detection of human IgG and IgM,using highly purified antibody calibrators (standards). Antibodies werestored at 4° C. as sterile microfiltered solutions.

Example 7 Synthesis of Peptide Conjugates with BSA, StrAv and HRP

Solutions of Maleimide-BSA (50–100 mg/ml), Maleimide-StrAv (5–7 mg/ml)and Maleimide-HRP (11–15 mg/ml) in 0.1 M sodium phosphate, 1 mM EDTA, pH6.5, were mixed at appropriate molar ratio (in range of 0.5:1 to 8:1)with freshly prepared peptide solution in water (10 mg/ml). Molar ratiopeptide/protein was calculated on the basis of peptide concentrationadjusted for the content of reactive thiol groups determined in DTNBtest (40–80% of total peptide). For C6 peptide (MW 2,780) 1:1 molarratio was equivalent to 46.3 ug/mg BSA or StrAv (MW 60,000 for eitherprotein) and 69.5 ug/mg HRP (MW 40,000). For C10 peptide (MW 1,241) 1:1molar ratio was at 20.6 ug/mg BSA or StrAv and 31 ug/mg HRP. Mixtureswere incubated for 30–60 min at room temperature. Almost 100% ofthiol-peptides reacted with maleimide, as judged by DTNB test ofreaction mixtures. Then, solution of beta-mercaptoethanol, 10 mg/ml inwater, was added at 3–5-fold molar excess over residual maleimidegroups. Conjugates were purified by dialysis against 0.1M sodiumphosphate, 1 mM EDTA, pH 6.5 using dialysis membrane with cut-off12,000–14,000 Da (to remove the fraction of unconjugated peptides thatdo not have reactive thiols). After dialysis, protein concentration wasdetermined using A₂₈₀ or A₄₀₃ coefficients shown in the example. Peptideconcentration in conjugate solutions was calculated assuming that alladded peptide with reactive SH groups has reacted withmaleimide-activated proteins (as confirmed by DTNB test), using thevalues of weigh ratios shown above.

Example 8 Synthesis of Peptide-BSA-HRP Conjugates

Peptide-BSA-HRP conjugates were prepared using peptide-BSA conjugatessynthesized as described above, but without inactivation of residualmaleimide groups with beta-mercaptoethanol. Peptide-BSA conjugates weremixed with thiolated HRP solution containing approx 1.2 SH groups perHRP molecule, prepared as described in Example 1. Molar ratios HRP/BSAwere 1:1, 2:1, 3:1 and 4.5:1, based on weight ratio of 0.66 mg HRP permg BSA for 1:1 ratio. Mixtures were incubated for 30 min, then solutionof N-ethylmaleimide (1 mg/ml in water) was added in amount equivalent toamount of original thiol content in SH-HRP. Mixtures were incubated 10min more, and finally residual maleimide groups were inactivated byaddition of beta-mercaptoethanol solution (10 mg/ml in water), in slightexcess to total amount of maleimide groups in reaction mixture. Theconcentration of peptides in conjugates was calculated from amount ofpeptides added into reaction as a peptide-BSA conjugate.

Example 9 Plate Coating Procedures for Bi-BSA, Bi-PEG-BSA, Peptide-BSA,StrAv-biotin-peptide

Polystyrol plates (Costar HB) were coated with capture reagents fromPBS-sodium azide overnight at 4–8° C. Bi-BSA and Bi-PEG-BSA (0.5–4ug/ml) were adsorbed at 200–250 ul/well, peptide-BSA reagents (0.5–2 ugBSA/ml) and StrAv (1–2 ug/ml) were adsorbed at 100 ul/well. Forblocking, PBS-0.05% Tween-20 was used for plates with biotinylated BSA,and 1% Casein-4% sucrose, pH 8.0, was used for plates with peptide-BSAor peptide-StrAv. Biotinylated peptides were immobilized on platescoated with StrAv at 0.5 ug/ml (100 ul/well) in the casein-sucroseblocking buffer. Plates were dried at room temperature overnight andstored in bags with desiccant.

Example 10 Adsorption of Capture Reagent on NC Membranes

Biotin-PEG-BSA was applied on dicks of NC membrane of 0.45 um or 1.2 umpore size (Schleicher&Schuell) installed into filtration device.Solution was prepared in PBS-azide with concentration of BSA 0.05–0.2mg/ml, and 0.5 ml was used per 14-mm diameter disc. After aspiration ofapplied aliquots, discs were washed two times with 1.0 ml of PBS-0.05%Tween-20.

Biotin-PEG-BSA was striped on NC membrane (0.45 um) using Bio-Dotdispenser from PBS-azide solution at concentration 0.2–1.0 mg/ml at pumpflow rate 2 ul/cm and platform speed 50 mm/sec. Membranes was dried30–60 min at room temperature, blocked in PBS-0.05% Tween-20 for 1 hour,and dried again.

Example 11 Preparation of C6-BSA Colloidal Gold Conjugates

Colloidal gold particles (30 nm size) were prepared using standardcitrate method (De Mey, 1986), pH was adjusted to 7.5 with 0.2 Mpotassium carbonate. Colloidal gold/C6-BSA conjugates were prepared fromC6-BSA conjugates synthesized as described in Example, withbeta-mercaptoehanol treatment to inactivate residual maleimide groups.C6-BSA conjugates were adsorbed at 0.5–2 ug/ml, and then blocked usingPEG 15,000/BSA mixture. Amount of C6-BSA used for adsorption wasslightly less than the maximum protecting concentration. Amount of boundC6-BSA was calculated assuming that all added conjugate was bound togold particles using OD518 nm values as a measure of colloidal goldconcentrations.

Example 12 Basic Format for SICS-DLA ELISA (Antibody Assay in SICS-DLAELISA)

Test samples (0.05 ml), which may be undiluted or diluted sera, purifiedanti-peptide antibodies in various diluents (normal human or calf serum,PBS with casein-Tween-20), were added into wells of plates coated withbiotinylated BSA as described in Example 5. Conjugate solution (0.05 ml)containing mixture of Peptide-StrAv and Peptide-HRP or Peptide-BSA-HRP(range of peptide concentration 0.01–0.16 ug/ml) in HRP/ProteinStabilizer (SurModics, Inc) was added. Plates were incubated withagitation on plate shaker/agitator for 25 min and washed four times withPBS-Tween-20. TMB substrate solution (Moss, Inc) was added (0.1 ml) andplates incubated for 4 min with agitation. Stop solution (0.1 ml of 0.1N sulfuric acid) was added and absorbance at 450 nm was determined inELISA plate reader blanked on air.

FIG. 1 shows the results. Highest sensitivity was obtained at ratio 0.5Mol C6 peptide:1 Mol StrAv (0.5:1) and C6-HRP conjugates at ratio 1 MolC6 peptide: 1 Mol HRP (1:1). The concentration of C6 peptide inconjugates mixture was 0.03 ug/ml for C6-StrAv conjugates and 0.05 ug/mlfor C6-HRP conjugates. The figure insert demonstrates linearity in thedynamic range of the ELISA reader. The detection limit, calculated asantibody concentration at OD=Blank OD+50%=30 ng/ml. The total assay timewas approximately 35 min.

FIG. 12 shows scatter plots of OD for a panel of normal blood donors(412 samples) tested in parallel in conventional IgG/IgM C6 ELISA (atdilution 1:20) with C6 peptide immobilized on solid phase and new teston undiluted sera as described in above. Very low OD values obtained forthese normal samples in the new test were practically indistinguishablefrom the OD of the blank. Cutoff values were calculated as average ODplus 3 standard deviations. The cutoff value for the new test was 0.11and 0.35 for conventional test.

Example 13 Basic Format for Conventional ELISA Using Anti-human IgG/IgMConjugates

Test samples (0.1 ml) containing 1:20 diluted sera in casein-Tween-20diluent were added into plates coated with peptide-BSA conjugates orbiotinylated peptides through StrAv bridge as described in Examples 9.Plates were incubated still at room temperature for 30 min and washedthree times with PBS-Tween-20. Conjugate solution (0.1 ml) containinggoat anti-human IgG, IgM or IgG/IgM (Jackson Immunoresearch Lab) at 0.16ug/ml in StabilZyme HRP (SurModics, Inc) was added. Plates wereincubated for 20 min at room temperature and washed four times. TMBELISA substrate (0.1 ml) was added and plates were incubated for 4 minbefore stopping with 0.1 ml of 0.1 N sulfuric acid. Absorbance at 450 nmwas measured in plate reader blanked on air.

Example 14 Analysis of Competition Between Immobilized Peptides andPeptides in Solution for Binding to Anti-peptide Antibodies

Plates were coated with peptide-BSA conjugates at molar ratiopeptide-BSA of 2:1 as described in Example 7. Samples containing knownamount of anti-peptide antibodies (in a range 0.1–1.6 ug/ml) were firstpre-incubated in separate tubes for 20 min with serially dilutedpeptides or peptide conjugates (in a peptide concentration range0.006–280 ug/ml). Than, 0.1 ml aliquots were transferred into platescoated with peptide conjugates. Plates were incubated for 30 min andwashed three times. Conjugate solution containing human IgG- orIgM-specific conjugate was added as described in Example 13. Furthersteps were as in Example 13.

Example 15 Procedure for Flow-through Test with HRP Label

Samples (200 ul) were prepared from microfiltered Positive control serumof IgG/IgM C6 ELISA test (Immunetics) by dilution with normal calfserum. Equal volume of C6 conjugate mixture (C6-StrAv, 0.5:1, 0.03ug/ml, and C6-HRP, 1:1, 0.05 ug/ml) was added to samples. Afterincubation for 5–10 min, the sample-conjugates mixture was applied ondiscs of NC membrane (0.45 um) coated with Bi-PEG-BSA as described inExample 9. After short incubation (1 min), liquid was filtered throughmembrane by applying positive pressure on the top. Membranes were washedthree times with 1 ml PBS-Tween-20. Then, 0.5 ml of TMB precipitatingmembrane substrate (Moss, Inc) was added. After 2 min, the substrate wasfiltered and membranes washed with water.

Example 16 Procedure for Flow-through Test with Colloidal Gold Label

Samples described in Example 15 were mixed with conjugates (C6-StrAv,0.5:1, 0.1 ug/ml and C6-BSA (2:1)-Gold, 0.05 ug/ml) and incubated for 30min with agitation. Mixtures were then applied on discs of NC membrane(1.2 um), coated with Bi-PEG-BSA as described in Example 10, installedinto filtration device and incubated for another 5 min before completionof filtration. Discs were washed two times with PBS-Tween-20.

Example 17 Procedure for Dot-blot Test with HRP Label

Samples (0.25 ml) were mixed with equal volume of C6 conjugates asdescribed in Example 15, and placed in a microtray. Strips of NCmembrane with applied Bi-PEG-BSA as described in Example 10 were placedinto sample-conjugate mixtures and incubated with agitation for 30 min.After complete aspiration of liquid, strips were washed three times with2 ml PBS-Tween-20 and once with water. TMB membrane ELISA substrate wasadded and strips incubated for 4 min with agitation. Finally, thesubstrate was aspirated and membranes rinsed with water.

Example 18 Procedure for Dot-blot Test with Colloidal Gold Label

Strips containing Bi-PEG-BSA lines were prepared as in Example 10 andwere placed into microtrays containing 0.5 ml sera samples prepared fromPositive control as in Example 15. A mixture of C6-StrAv and C6-BSA-Goldconjugates was added. Final concentration of C6 peptide was as describedin Example 16. Mixtures were incubated with agitation for 45 min, thanwashed with PBS-tween-20.

Example 19 Competitive Assay for Antigen Detection Using PeptideConjugates

Samples containing known amount of recombinant VLSe (0.045 ml) wereplaced into wells coated with Bi-PEG-BSA as described in Example 9.Solution of anti-C6 IgG (10 ug/ml was added at 0.05 ul/well. Mixtureswere incubated for 10 min with agitation and 0.05 ml of conjugatesolution containing 0.03 ug/ml C6-StrAv (0.5:1) and 0.05 ug/ml C6-HRP(1:1) was added. Plates were incubated with agitation for 25 min, washedfour times with PBS-tween-20, and bound HRP was detected as described inExample 13.

Example 20 Analysis of Antibody Classes Involved in Specific ImmuneComplex with Labeled Peptide Antigens

Samples of human patients sera (0.05 ul) were diluted in coated wellprepared as described in Example 12 with 0.045 ml calf serum (Sigma) orCasein-tween-20 diluent. Conjugate solution (0.05 ml) containing mixtureof peptide-StrAv plus peptide-HRP at concentration described in Example12 was added. The mixture were incubated for 25 min and agitation,aspirated and washed 3 times with PBS-tween. Solution of Goat-anti-humanIgG or IgM-HRP at concentration 0.08 ug/ml was added and plates wereincubated for 15 min at agitation. After washing (four times) bound HRPwas detected using standard procedure for TMB ELISA substrate asdescribed in Example 13.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims.

1. A kit comprising a first component comprising biotin linked, througha polyethylene glycol spacer, to albumin, a second component comprisinga biotin-binding moiety conjugated to an antigen or hapten that binds anantimicrobial antibody to be detected in a sample, and a third componentcomprising the antigen or hapten conjugated to a label.
 2. The kit ofclaim 1, wherein the second and third components are supplied as drysamples or liquid samples, wherein the dry or liquid samples may beseparate or mixtures of the second and third components.
 3. The kit ofclaim 1, wherein the second and third components comprise more than oneantigen or hapten molecule.
 4. The kit of claim 1, wherein the secondcomponent and the third component each comprise two or more differentantigens or haptens that are chemically linked to the biotin-bindingmoiety or the detector label, respectively.
 5. The kit of claim 1,wherein the biotin-binding moiety is avidin or streptavidin.
 6. The kitof claim 1, wherein the sample is taken from a human or animal having amicrobial infection.
 7. The kit claim 6, wherein the microbial infectionis Lyme disease.
 8. The kit of claim 1, wherein the antigen is C6peptide derived from VLSe protein of B. burgdorferi.
 9. The kit of claim1, wherein the antigen is C10 peptide derived from OspC protein of B.burgdorferi.
 10. The kit of claim 1, wherein the antigens are peptidesderived from VLSe protein (C6) and OspC protein (C10) of B. burgdorferi.11. The kit of claim 1, further comprising instructions for practicingthe method of detecting an antibody in a sample.
 12. A kit comprising asolid support to which is noncovalently adsorbed a first componentcomprising biotin linked, through a polyethylene glycol spacer, toalbumin; a second component comprising a biotin-binding moietyconjugated to an antigen or hapten that binds an antimicrobial antibodyto be detected in a sample; and a third component comprising the antigenor hapten conjugated to a label.
 13. A kit of claim 12 wherein thesecond and third components are present in a single reagent mixture.